During the past decade the all-enzymatic hydrolysis of starch to syrups has gained wide and steadily increasing acceptance within the starch processing industry. On a world-wide basis the present enzymatic production of dextrose syrup from starch is estimated to exceed 3 million tons (calculated as dry substance) per annum compared with about 0.4 million tons ten years ago.
The process generally adopted for enzyme--enzyme hydrolysis of starch encompasses the sequential steps of liquefaction and saccharification, the former being catalysed by an alpha-amylase, such as the thermostable B. licheniformis alpha-amylase, e.g. TERMAMYL.RTM. supplied by NOVO Industri A/S, Denmark, and the saccharification to dextrose (D-glucose) being effected in the presence of a glucoamylase, usually of fungal origin, such as AMG-150 L, also obtainable from the above named company. Obviously, the dextrose syrup producer aims at obtaining the highest possible yield of dextrose with the least possible expenditure of enzymes and of energy.
The highest dextrose level attainable by the conventional process, starting with 30-40 percent (by weight) starch suspension and saccharifying at 30 percent dry solids (D.S.), is about 96 percent (by weight) dextrose (96 DX). The reasons why the conventional starch conversion process does not proceed appreciably beyond that limit are essentially twofold.
Firstly, amylopectin (which constitutes about 80% of the majority of industrially important starches, including that of corn or maize), exhibits a branched chain structure in that it contains a significant number of alpha-1,6-glycosidic bonds. Whereas amylopectin is only partially degraded by alpha-amylase because alpha-amylase is practically devoid of alpha-1,6-glucosidase activity, substantial hydrolysis of the branched oligosaccharides including alpha-limit dextrins occurs in the subsequent saccharification step catalyzed by glucoamylase which also hydrolyses alpha-1,6-glycosidic links. However, the latter reaction proceeds at a considerably lower rate than the corresponding hydrolysis of alpha-1,4-bonds, whereby complete saccharification is impeded. Attempts to remedy the situation by adding more glucoamylase collides with a second obstructive feature (apart from incurring higher enzyme costs), namely the ability of glucoamylase also to catalyze dextrose polymerization (the so-called reversionary reaction).
It should be mentioned in passing that an increase of starch conversion from about 96 DX to about 98 DX (which for certain uses of dextrose is regarded as a highly significant improvement) entailing thereby a reduction in the content of non-dextrose contaminants by about 50 percent, can be achieved by employing a relatively high level of glucoamylase combined with a dilution of the substrate to about 15 percent D.S., vide U.S. Pat. No. 4,017,363. However, the subsequent concentration of such a dextrose solution to the higher conventional dry solids levels is energy consuming.
The prior art has suggested employment of glucoamylase and a debranching enzyme simultaneously to obtain a significant increase in dextrose level, the rationale being that debranching enzymes have been shown to efficiently hydrolyse specific types of alpha-1,6-glycosidic bonds occurring in branched chain oligosaccharides, and certain alpha-limit dextrins. In this respect reference is made to U.S. Pat. No. 3,897,305 disclosing the combined use of glucoamylase and Aerobacter aerogenes (Klebsiella pneumoniae) pullulanase, whereby a significant increase in DX of up to 2 percent can be achieved for syrups containing at least 30 percent D.S. Similar results have been demonstrated for the combined action of glucoamylase and another debranching enzyme, viz. Pseudomonas amyloderamosa isoamylase as described in British Patent Application No. 8107287.
However, in the first instance practically no saving of glucoamylase is achieved because the pH optimum of K. pneumoniae pullulanase makes it mandatory to conduct the saccharification at a relatively high pH (5,5-6) whereat the activity of glucoamylase is dramatically reduced.
The same problem is not encountered with the isoamylase which has a pH optimum much closer to that of glucoamylase, whereby the dosage of the latter can be substantially reduced (by about 50 percent), simultaneously with the attainment of an increase in DX value of 1-2 percent. However, a serious drawback of the isoamylase process (and actually shared by the known pullulanase process as well) is the heat lability of the debranching enzymes known in the art. This has meant that heretofore no saccharification in the presence of debranching enzyme has been technically feasible above about 55.degree. C., whereas glucoamylase per se is adequately stable even at 60.degree. C. at which temperature level the risk of microbial contamination of substrates is significantly reduced as compared with lower temperatures.
Obstacles akin to those described hereinbefore have been encountered in the conversion of starch to high maltose syrup by means of beta-amylases. Like the alpha-amylases, beta-amylases are only capable of partially degrading amylopectin, in that hydrolysis thereof stops as an 1,6-alpha-branch point is approached. By combining the action of beta-amylase with that of a debranching enzyme, such as pullulanase or isoamylase, a substantial increase in maltose content can be achieved as disclosed in British Pat. No. 1.144.950 and U.S. Pat. No. 3,677,896. However, again saccharification temperatures above 55.degree. C. are not feasible due to the heat lability of the debranching enzymes, whereby the risk of bacterial contamination is substantially increased.
It is an object of the present invention to obviate the shortcomings of the debranching enzymes known heretofore by furnishing a novel debranching enzyme having a temperature stability comparable to that of glucoamylase, and furthermore, possessing a pH optimum close to that of glucoamylase.
The invention resides in the surprising discovery that a novel debranching enzyme of the pullulanase type having such properties is produced by newly discovered microorganisms of the genus Bacillus belonging to the taxonomic group as hereinafter defined.